The present invention relates to a sulfated fucogalactan which is useful as a reagent for glycotechnology, a smaller molecule from the sulfated polysaccharide and a method for producing the same, as well as a sulfated fucogalactan-digesting enzyme which is useful in a field of glycotechnology and a method for producing the enzyme.
Brown algae contain a variety of sulfated-fucose-containing polysaccharides. For example, sulfated-fucose-containing polysaccharides such as (1) sulfated fucans consisting of fucose and sulfate groups; (2) sulfated fucoglucuronomannans containing glucuronic acid, mannose, fucose and sulfate groups, e.g., the sulfated-fucose-containing polysaccharide-U as described in WO 97/26896 (approximate molar ratio of constituting saccharides, fucose: mannose: galactose: uronic acid: sulfate group=10:7:4:5:20; hereinafter referred to as U-fucoidan); and (3) sulfated fucogalactan consisting of fucose and galactose, e.g., the sulfated-fucose-containing polysaccharide-F as described in WO 97/26896 (approximate molar ratio of constituting saccharides, fucose: galactose=10:1; hereinafter referred to as F-fucoidan) are known. Almost all of these sulfated-fucose-containing polysaccharides are macromolecular anions. Therefore, they behave in a chemically and physically similar manner in various purification steps, making it difficult to separate them each other. For this reason, biological activities of sulfated-fucose-containing polysaccharides derived from brown algae have often been examined without separating them each other. Therefore, it was difficult to identify the sulfated-fucose-containing polysaccharide that was responsible for the observed biological activity.
To date, correlation between an activity and a molecule has been known for the sulfated fucan fraction as described in Agricultural and Biological Chemistry, 44(8): 1965-1966 (1980), which is responsible for anticoagulant activity, and U-fucoidan as described in WO 97/26896, which is responsible for an apoptosis-inducing activity against tumor cells.
The use of the sulfated fucan fraction as an anticoagulant in place of heparin has been examined. However, if the sulfated fucan is to be used as a pharmaceutical, it is required to obtain a highly pure sulfated fucan in order to avoid side effects due to unexpected activities. Thus, a method therefor has been desired.
Regarding U-fucoidan, it is similarly required to conveniently obtain a highly pure sulfated-fucose-containing polysaccharide-U in order to prepare a pharmaceutical utilizing the apoptosis-inducing activity against tumor cells. Thus, a method therefor has been desired.
Generally, enzymatic digestion is the most efficient manner utilized for structural analysis of polysaccharides and production of oligosaccharides. Furthermore, only one polysaccharide can be readily removed from a mixture of polysaccharides which are hardly separated each other as follows. The polysaccharide to be removed is converted into smaller molecules by digesting it with an enzyme that specifically digests the polysaccharide. The mixture is then subjected to molecular weight fractionation such as ultrafiltration.
If an enzyme that specifically digests a sulfated fucogalactan is available, structural analysis of the sulfated fucogalactan and production of a sulfated fucogalactan oligosaccharide are readily conducted.
As described above, a sulfated fucogalactan-digesting enzyme and a method for enzymatically producing a sulfated fucogalactan oligosaccharide have been desired.
Thus, the main object of the present invention is to provide (1) a sulfated fucogalactan or a salt thereof which is useful as a reagent for glycotechnology or an inducer of hepatocyte growth factor (HGF) production; (2) a smaller molecule obtained by allowing a sulfated fucogalactan-digesting enzyme to act on a sulfated fucogalactan, or a salt thereof; (3) a sulfated fucogalactan-digesting enzyme which is useful for glycotechnology; (4) a method for producing a smaller molecule obtained by allowing said enzyme to act on a sulfated fucogalactan or a salt thereof; and (5) a method for producing a sulfated fucogalactan-digesting enzyme.
As a result of intensive study, the present inventors have found a sulfated fucogalactan contained in brown algae, a sulfated fucogalactan-digesting enzyme that digests the sulfated polysaccharide and a method for producing the same. Furthermore, the present inventors have found a smaller molecule from a sulfated fucogalactan which can be utilized as a reagent for glycotechnology and a method for producing the same, thereby completing the present invention.
The first aspect of the present invention relates to a sulfated fucogalactan having the following chemical and physical properties or a salt thereof. The sulfated fucogalactan contains galactose and fucose as constituting saccharides at a molar ratio of 1:1 to 6:1 and contains a sulfated saccharide of general formula (XI) as an essential component of the constituting saccharides: 
wherein R is H or SO3H.
Furthermore, the sulfated fucogalactan is converted into smaller molecules by a sulfated fucogalactan-digesting enzyme of the present invention to generate at least one compound selected from the group consisting of the compounds of general formulas (I) to (IV): 
wherein R is H or SO3H.
The second aspect of the present invention relates to a saccharide having a chemical structure selected from the group consisting of general formulas (II), (IIIxe2x80x2) and (IV): 
wherein R is H or SO3H, or a salt thereof.
The third aspect of the present invention relates to a sulfated fucogalactan-digesting enzyme having the following chemical and physical properties. The enzyme can act on a sulfated fucogalactan containing galactose and fucose as constituting saccharides at a molar ratio of 1:1 to 6:1 or a salt thereof to convert the sulfated fucogalactan into smaller molecules and generate an oligosaccharide having sulfated galactose or galactose at its reducing end, has an optimal pH of about 7 to 9, and has an optimal temperature of about 25 to 45xc2x0 C.
The fourth aspect of the present invention relates to a method for producing a smaller molecule from a sulfated fucogalactan or a salt thereof, characterized in that the method comprises allowing the sulfated fucogalactan-digesting enzyme according to the third aspect to act on a sulfated fucogalactan derived from brown algae or a salt thereof and obtaining a smaller molecule. Examples of the smaller molecules obtained using the enzyme include the oligosaccharide or a salt thereof according to the second aspect.
The fifth aspect of the present invention relates to a method for producing the sulfated fucogalactan-digesting enzyme according to the third aspect, characterized in that the method comprises culturing a bacterium of genus Flavobacterium capable of producing the sulfated fucogalactan-digesting enzyme and collecting the enzyme from the culture.